This project is primarily an investigation of four low molecular weight proteinase inhibitors which are found in potato tubers. One of these inhibitors is unusual in that it is specific for the pancreatic carboxypeptidases, whereas two of the others affect chymotrypsin and the fourth inhibits trypsin. The proposed research involves a complete sequence determination of these inhibitors. The mechanism of action of each inhibitor will be probed by chemical modification, reactive site cleavage, and crystallization of enzyme-inhibitor complexes in preparation for X-ray diffraction analysis. A second aspect of the proposed research is the preparation of highly specific affinity labels for target proteinases by the selective introduction of reactive groups onto inhibitor molecules. The affinity label analogs and target enzymes will be incubated, and the presence of covalent enzyme-inhibitor adducts will be detected by SDS-polyacrylamide gel electrophoresis. The final aspect of the work is the elucidation of the roles which proteinases play in such medically relevant phenomena as natural killer lymphocyte activity and tumor cell invasion and metastasis. Initially several proteinases inhibitors, including those from potato tubers, will be tested for the ability to interfere with these processes. Inhibitors shown to be effective will be immobilized, and the immobilized inhibitors will be used to purify the critical target enzymes, which will then be characterized.